S164: CLONAL DYNAMICS, IMMUNE PHENOTYPES, AND TARGETS OF BONE MARROW-INFILTRATING T CELLS IN ACQUIRED APLASTIC ANEMIA
Authors: A. Ben Hamza, C. Welters, M. Brüggemann, K. Dietze, L. Bullinger, T. H. Brümmendorf, J. Strobel, H. Hackstein, K. Dornmair, F. Beier, L. Hansmann
Journal: HemaSphere (2022)

Background: Acquired aplastic anemia is a hematological disease characterized by hypocellular bone marrow with insufficient hematopoiesis and pancytopenia. High response rates to immunosuppressive therapy with anti-thymocyte-globulin and cyclosporin A suggest a key role for T cells in disease pathogenesis. However, bone marrow-infiltrating clonal T cell expansion, associated immune phenotypes, and T cell targets remain poorly understood. Aims: To defined clonal T cell expansion, associated immune phenotypes, and potential T cell targets in bone marrow specimens of aplastic anemia patients before and after immunosuppressive therapy. Methods: Within bone marrow of 15 patients with acquired aplastic anemia, we determined T cell clone-associated immune phenotypes by multi-parameter flow cytometry single cell index sorting for subsequent T cell receptor (TCR) αβ and cytokine/transcription factor sequencing. T cell clones of 10 patients were monitored before and after immunosuppressive therapy (median time span: 9 months) by TCRβ repertoire sequencing. Twenty-seven TCRs of expanded clones were re-expressed in reporter cell lines to determine recognition of autologous hematopoietic precursor cells. Results: We detected oligoclonal T cell expansion in all patients and 92.9% of all expanded clones were CD8+, while only 9.6% were CD4+. Frequencies of dominant T cell clones varied between patients (1.1-20.6% of CD8+ T cells). Expanded CD8+ clones were almost exclusively CCR7- PD1- TIM3- CD39- and frequently expressed cytokines and transcription factors associated with cytotoxic effector differentiation (IFNG, GZMB, PRF1, TBX21). More than 50% of the CD45RA+ clones (TEMRA) were CD57+, while CD45RA- clones (TEM) were mainly CD28+ and smaller in size. While TEM clones showed similar frequencies in bone marrow and peripheral blood, TEMRA displayed higher localization-dependent differences in size. Interestingly, almost all (91.1%) expanded clones with frequencies >1% of all T cells persisted in the bone marrow after immunosuppressive therapy independent of clinical response. Similarly, highly expanded CD4+ clones, albeit low in numbers, were stable in frequencies before and after therapy. To determine targets of CD8+ T cell clones with strong expansion, we re-expressed twenty-seven TCRs of eight patients and found five to be specific for immunodominant epitopes of cytomegalovirus. Strikingly, two TCRs recognized hematopoietic progenitor cells expanded from CD34-enriched autologous bone marrow. Summary/Conclusion: We tracked T cell clonotypes and associated immune phenotypes under immunosuppressive therapy at the single cell level. Expanded CD8+ clones showed cytotoxic effector differentiation and persisted in the bone marrow after immunosuppressive therapy even in situations of therapeutic response, challenging the hypothesis that clinical response follows the decline of highly expanded autoreactive T cell clones. We identified two expanded T cell clones that recognized hematopoietic progenitor populations providing experimental proof of concept that expanded autoreactive T cell clones can be critically involved in aplastic anemia pathogenesis. FB and LH contributed equally.